Project Summary Coronary artery disease (CAD) is a leading cause of death worldwide. A major causal risk factor for CAD is elevated low-density lipoprotein cholesterol (LDL-C) levels. While reduction in LDL-C is a cornerstone of the prevention and treatment of CAD, many patients continue to experience CAD events despite highly effective LDL-C reduction, indicating important residual risk. Plasma triglycerides (TGs) are an independent predictor of CAD risk. TGs are strongly associated with CAD events even in statin-treated patients with low LDL-C levels, and are one of the strongest predictors of on-statin vascular risk. TGs are carried in TG-rich lipoproteins (TRLs). TRLs provide energy to extrahepatic tissues through the activity of cell-surface lipoprotein lipase (LPL), which hydrolyzes TGs in TRLs for the local absorption of the released free fatty acids. Recent large-scale human genetics studies have established that genetic variants associated with TG levels are also strongly associated with CAD. These genetic studies have especially pointed to the LPL pathway, and the apolipoprotein ApoA-V has been identified as a particularly interesting modulator of LPL. ApoA-V is primarily secreted from the liver and can exchange between high-density lipoproteins (HDLs) and TRLs. ApoA-V has been shown to enhance LPL activity, although the precise mechanism remains unclear. Human genetics have strongly supported ApoA-V?s role in TG metabolism: case-control, family-based sequencing studies, and an exome sequencing study have implicated several coding variants in hypertriglyceridemia (hyperTG), hyperchylomicronemia, and early myocardial infarction, respectively. Additionally, the hyperTG phenotype in apoa5 knockout mice can be suppressed by recombinant human ApoA-V or human APOA5 AAV. My work seeks to functionally characterize the effects of selected natural APOA5 variants, both in vitro and in vivo. Preliminary evidence supports 2 predicted loss-of-function (LoF) variants Q275X and Q305X and 3 ambiguous and/or potentially beneficial variants (predicted gain-of-function (GoF)) D37E, P215L, and T292I, that may be particularly informative for elucidating ApoA-V function. My first goal is to assess lipoprotein binding and LPL activity enhancement of selected APOA5 variants, as ApoA-V?s ability to bind lipoproteins is critical to localizing the protein at the TRL-LPL interface. I hypothesize that APOA5 predicted LoF variants decrease lipoprotein binding, blunt LPL activation, and increase plasma TG levels, while APOA5 predicted GoF variants increase lipoprotein binding, resulting in reciprocal phenotypes. My second goal is to determine APOA5 variant impacts on long-term TG metabolism and atherosclerosis. I hypothesize that atherosclerotic mouse models expressing APOA5 predicted LoF variants will have decreased TG clearance and augmented atherosclerotic progression while APOA5 predicted GoF variants will have reciprocal phenotypes. The proposed studies will provide substantial new insight into ApoA-V?s modulation of plasma TGs, a targetable CAD risk factor, that could guide development of rationally-designed therapeutics.